Nanopore Biosensors For Sensitive Detection Of Drug Molecules Based On Cyclodextrin Adapter

Recently,the problem of drug residues in the entire food chain has become much more serious to human health.Therefore,establishment of efficient and rapid methods to detect drug residues has become one of the hotspots in the field of analytical chemistry,and how to improve the accuracy and sensitivity of detection methods is the priority of the research.Here a biosensor（（M113R）₇-am₇βCD）was constructed by using the mutantα-hemolysin protein（M113R）₇ as nanopore channel and 6-amino-6-deoxy-β-cyclodextrin（am₇-βCD）as adapter.A new method of drug detection was proposed and successfully applied into the detection of antituberculosis drug isoniazid（INH）and antibiotic drug ampicillin sodium salt（Amp）.This research provides a reference for the detection of other drug residuals,and it also extends the application of nanopore single molecule technology.The main contents are included as follows:⑴Reviews.The development history and basic principles of nanopore single molecule detection technology were briefly described.The application of nanopore detection based on adapters,especially cyclodextrin adapters,in the field of DNA sequencing,metal ions or small organic molecules detecting,and host-guest kinetics were highlighted.⑵Nanopore detection of isonizid molecule based on cyclodextrin adapter A biosensor of（M113R）₇-am₇βCD was constructed on the nanopore single-molecule level.The drug molecule isoniazid was detected based on the mentioned biosensor by the selection of proteins,cyclodextrin adapters,and the optimizing of experiment conditions（voltage,pH etc.）.The results showed that high voltage and neutral conditions can improve the detection sensitivity.The event frequency（f）,association rate constant(k_on)and formation constant（K_f）continuously increased as the voltage rised.The background signal of am₇βCD is much cleaner and the characteristic signal with isoniazid is more obvious in buffer solution of pH 8.0 compared with pH 6.0 and pH 10.0.The linear range and the detection limit of the sensor were 0.5-30μM and 2 nM,and it proved the established sensor has good selectivity by detecting common drug excipients and concomitant metal ions（above 40 times of isoniazid concentration）.⑶Nanopore detection of isoniazid based on cyclodextrin adapter The antibiotic ampicillin sodium salt was successfully tested based on the biosensor of（M113R）₇-am₇βCD.The interaction between cyclodextrin adapter and ampicillin sodium salt was investigated by calculating the kinetic constants.It was proved that the sensor we established has specific recognition for ampicillin sodium salt after the detection of other antibiotics such as gentamicin,doxorubicin,etc..The study indicated that the liner range and the detection limit of the sensor were 0.01-40μM and 8 nM,respectively.